Pathway Analysis of DEGs Using MapMan
Metabolic pathways analysis of DEGs was performed through MapMan to determine the effects of drought stress in root tissues of soybean. In our data, out of all up- and down-regulated DEGs of DTP and DSP, only 121 and 105 DEGs, respectively were mapped, and out of them only 99 and 87 were shown in Fig. 7B , whereas only 123 and 123 DEGs were mapped out of all up- and down-regulated DEGs of DTL and DSL, respectively, and 101 and 101were presented in the Fig. 7A by using Gmax_AFFY_09.m02 (Thimm et al., 2004). Comprehensive overview of DEGs, such as peroxidases, TFs, secondary metabolites, PRs, cell wall, signaling, hormone signaling, redox state etc. are presented inFig. 7A&B . Both MapMan and GO analysis are in well agreement, as in both cases DEGs enrichment were found similar in pathways and functional groups. However, most of the DEGs were assigned to specific functional groups, genes relevant to the terms hormone signaling, cell wall, antioxidant activity, signaling, proteolysis, TFs (MYB, ERF, WRKY, bZIP, and bHLH) were highly expressed in tolerant genotypes, and reverse trend was observed in sensitive genotypes.
Hence, consistency of GO and MapMan analyses revealed that drought stress response was highly enriched in the DEGs associated with water transport (GO:0006833), cell wall (GO:0005618), plant-type vacuole membrane (GO:0000325), antioxidant activity (GO:0016209), peroxidase activity (GO:0004601), catalytic-activity (GO:0003824), auxin transport (GO:0060918), protein-kinase activity (GO:0004672), secondary metabolite synthesis (GO:0044550), transcription-factor activity (GO:0003700), hormone-signaling (GO:0009755). Hence, these terms can play vital role in regulating drought stress tolerance at germination stage in soybean.
KEGG Pathway Analysis of DEGs Involved in Secondary Metabolism and Hormone-Mediated Signaling
Based on the GO and MapMan analysis as well as previous literature, secondary metabolites and hormone-mediated signaling plays important role in drought tolerance. Hence, DEGs related to secondary metabolites and hormone-mediated signaling were further subjected to KEGG pathway enrichment analysis. Our results showed that genes associated with biosynthesis of secondary metabolites such as shikimate acid, alkaloid, anthocyanin, lignin, flavonoids and terpenoid were mainly differentially expressed under drought stress in root tissues (Table 2). The number of up- and down-regulated genes associated with secondary metabolites biosynthesis such as shikimate acid, alkaloid, anthocyanin, lignin and terpenoid in both tolerant and sensitive genotypes are presented (Fig. S1; Table S8).
Furthermore, many DEGs related to AUX, ABA, GA, JA, ETH and Brassinosteroid (BR) were found in drought stressed soybean roots. The changes in the gene expressions of hormone‐responsive genes, and their regulating TFs are shown in Table 3. To further understand the regulation of phytohormone in soybean transcriptome, several DEGs related to the biosynthesis of phytohormones viz., AUX, ABA, GA, JA, ETH and BR were identified under drought stress in the soybean root tissues. The changes in the gene expressions of hormone‐responsive genes, and their regulating TFs are presented (Table 3; Fig. S2) . The number of DEGs involved in the hormonal-signaling were mostly related to AUX followed by GA, ETH and ABA (Table S13). This indicates important role of these hormones especially AUX in drought stress response at germination stage.